CN1234855C - Recombinant Flt3 ligand gene and its fusion gene and product - Google Patents

Recombinant Flt3 ligand gene and its fusion gene and product Download PDF

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Publication number
CN1234855C
CN1234855C CN 01132280 CN01132280A CN1234855C CN 1234855 C CN1234855 C CN 1234855C CN 01132280 CN01132280 CN 01132280 CN 01132280 A CN01132280 A CN 01132280A CN 1234855 C CN1234855 C CN 1234855C
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Prior art keywords
gene
flt3
sequence
ser
flt3 ligand
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CN1421529A (en
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王皓
高虹
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Three country Kin Pharmaceutical (Shanghai) Limited by Share Ltd
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ZHONGXIN GUOJIAN PHARMACEUTICAL CO Ltd SHANGHAI
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Abstract

The present invention provides a recombinant Flt3 ligand of which the codon is suitable for CHO cell expression. The present invention also provides a fusion gene comprising the recombinant gene. When the gene or the fusion gene of the present invention is in expression, the expression amount in mammalian cells such as CHO, etc. and the affinity to Flt3 are obviously improved.

Description

Reorganization Flt3 ligand gene, and fusion gene and product
Invention field
The present invention relates to the recombination of Flt3 part (c-fms sample casein kinase 3 parts), and the fusion gene and the product thereof that contain this recombination.
Background of invention
The Flt3 part has very unique biological purposes, and it is early stage hemopoieticgrowth factor, acts on the original hematopoietic stem of expressing the Flt3 acceptor, makes it propagation and differentiation, and suppresses their apoptosis, is the agonist and the protective material of hemopoietic stem cell.Its natural gene sequence is determined, and has obtained GenBank accession number T03858.Recently studies show that, the Flt3 part can also increase dendritic cell (Dendritic cells significantly, DC) and natural killer cell (natural killer cells, NK) quantity and function, because DC and NK play important effect in the immunological surveillance of body and immunoregulation, so the Flt3 part has good application prospects aspect antineoplastic immune.
The keying action in organism immune response in view of DC and NK, therefore the Flt3 part is expected to be applied in the tumor treatment, and its validity has obtained proof in a plurality of experimentation on animalies such as leukemia, fibrosarcoma, melanoma, lymphoma, prostate cancer, breast cancer, lung cancer.Whole body is used the Flt3 ligandin of reorganization, all can cause that the part tumor regression or the speed of growth obviously slow down, and may excite the tumour-specific immune response in these different tumor experiment models.At present, main and other cytokine combined utilization of the recombinant protein of Flt3 part or carry out gene therapy have improved curative effect.
But, because malignant tumour is escaped the immunity monitoring of body by various mechanism, in the immunizing power that suppresses body, the abnormal antigen of self is by body identification and effectively offered to quicken the formation and development of tumour, and may to be the Flt3 part can not cause it to disappear fully and control one of reason of distant metastasis the tumour of some immunogenicity differences for this.Itself and tumor by local radiotherapy are united, can cause apoptosis of tumor cells, make postradiation cell become ideal cancer antigen slowly-releasing storehouse, DC can obtain the antigen of apoptotic cell and effectively offer, and stimulates specific C D8 +The hyperplasia of CTL, when disappearing, local tumor can reduce the lungs distant metastasis, thereby prolong the survival rate of highly metastatic reduced immunogenicity Lewis lung cancer model mice, proved the possibility of tumour traditional remedies combined utilization such as Flt3 part and radiotherapy first.
Therefore, Flt3 part as early stage hemopoieticgrowth factor, possessed unique and powerful raise immunity, its recombinant protein is used separately or with other cytokine associating whole body, or gene therapy all is proved to be and can produces significant antitumous effect, demonstrating the good clinical application prospect aspect the prevention of tumour and the treatment.Yet present technology still exists expression amount low, and Flt3 part and the little problem of Flt3 avidity can not satisfy this area needs growing to antineoplastic immune.
Summary of the invention
The object of the present invention is to provide a kind of Flt3 ligand gene of reorganization, it can improve the expression of this gene in mammalian cell.
The present invention also aims to provide a kind of fusion gene that contains this recombination, and the fusion rotein that obtains thus, this fusion rotein has stronger immunocompetence.
The present invention also aims to provide the recombinant vectors that contains recombination of the present invention, but transfection mammalian cell and realize high expression level.
In one embodiment of the invention, provide a kind of reorganization Flt3 ligand gene sequence, this reorganization Flt3 ligand gene has the nucleotide sequence of SEQ ID NO:3.
In another embodiment of the present invention, provide a kind of fusion gene, human IgG1's antibody FC (Fc, crystallizablefragment) gene that this fusion gene contains reorganization Flt3 ligand gene sequence and merges with it.In a preference of the present invention, this fusion gene also comprises one section joint sequence that is positioned between reorganization Flt3 ligand gene and the human IgG1's antibody Fc fragment, the aminoacid sequence of its dna sequence encoding SEQ IDNO:5.In another preference, the dna sequence dna of this joint is SEQ ID NO:4.
Also have among the embodiment of the present invention, a kind of recombinant vectors is provided, this recombinant vectors contains the above fusion gene described in the embodiment.
In another embodiment of the present invention, provide a kind of host cell, this host cell is by above-mentioned carrier transfection.
In one embodiment of the invention, provide a kind of protein, this protein contains Flt3 part and human IgG1's antibody Fc fragment.In a preference of the present invention, described protein also contains the aminoacid sequence SEQ ID NO:5 of joint peptide.
Term used herein " host cell " means and contains an expression vector and support the cell that this expression vector duplicates or expresses.Host cell can be prokaryotic cell prokaryocytes such as intestinal bacteria, or yeast, insect, batrachians or eukaryotic cells such as mammalian cell such as CHO, HeLa, as cultured cells, explant and cells in vivo.In a preference of the present invention, this host cell is CHO.
Term used herein " carrier " is reorganization or the synthetic nucleic acid construct that produces, and has the specific nucleic acid element that a series of permission specific nucleic acids are transcribed in host cell.Carrier can be the part of plasmid, virus or nucleic acid fragment.Usually, carrier comprises the nucleic acid to be transcribed that is connected with the promotor manipulative capability.
When pair cell or DNA, protein or carrier use term " reorganization ", refer to this cell, DNA, protein or carrier by introducing allogeneic dna sequence DNA or protein, or change n DNA or protein and modified, or should cell-derived cell from modification like this.Therefore for example, reconstitution cell has been expressed undiscovered gene in natural (non-reorganization) form of cell, or expresses and lowly or not express natural gene.
The accompanying drawing summary
Fig. 1 is the structural representation of pMG18.Wherein " hCMV pro " represents the human cytomegalic inclusion disease virus promotor.
Embodiment
According to the data (accession number T03858) of GenBank, the natural encoding sequence of Flt3 ligand gene is (SEQ ID NO:1):
acccaggact gctccttcca acacagcccc atctcctccg acttcgctgt caaaatccgt 60
gagctgtctg actacctgct tcaagattac ccagtcaccg tggcctccaa cctgcaggac 120
gaggagctct gcgggggcct ctggcggctg gtcctggcac agcgctggat ggagcggctc 180
aagactgtcg ctgggtccaa gatgcaaggc ttgctggagc gcgtgaacac ggagatacac 240
tttgtcacca aatgtgcctt tcagcccccc cccagctgtc ttcgcttcgt ccagaccaac 300
atctcccgcc tcctgcagga gacctccgag cagctggtgg cgctgaagcc ctggatcact 360
cgccagaact tctcccggtg cctggagctg cagtgtcagc ccgactcctc aaccctgcca 420
cccccatgga gtccccggcc cctggaggcc acagccccga cagccccc 468
Its aminoacid sequence is (SEQ ID NO:2):
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30
Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp
35 40 45
Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala
50 55 60
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His
65 70 75 80
Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95
Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu
100 105 110
Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125
Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140
Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
145 150 155
Wherein 23 amino acid of N-end are signal peptides.
In order to realize purpose of the present invention, the invention provides a kind of new reorganization Flt3 ligand gene, codon wherein meet mammalian cell access to your password the son preferences, the sequence of described recombination is (SEQ ID NO:3):
acccaggact gctccttcca gcactccccc atctcctccg acttcgccgt gaagatccgc 60
gagctgtccg actacctgct gcaggactac cccgtgaccg tggcctccaa cctgcaggac 120
gaggagctgt gcggcggcct gtggcgcctg gtgctggccc agcgctggat ggagcgcctg 180
aagaccgtgg ccggctccaa gatgcagggc ctgctggagc gcgtgaacac cgagatccac 240
ttcgtgacca agtgcgcctt ccagcccccc ccctcctgcc tgcgcttcgt gcagaccaac 300
atctcccgcc tgctgcagga gacctccgag cagctggtgg ccctgaagcc ctggatcacc 360
cgccagaact tctcccgctg cctggagctg cagtgccagc ccgactcctc caccctgccc 420
cccccctggt ccccccgccc cctggaggcc accgccccca ccgccccc 468
For the purpose of cloning and expressing, preceding 23 amino acid of this gene encoding production N-end are signal peptide sequences.
The present invention also provides the fusion gene that contains above-mentioned reorganization Flt3 ligand gene sequence, and wherein this recombination and human IgG1's Fc fragment gene merges.
In a kind of preferred implementation of the present invention, reorganization Flt3 ligand gene merges by one section joint sequence and human IgG1's Fc fragment, and its amino acid sequence coded is SEQ ID NO:5
Ser Ser Ser Ala Ala Ser Ser Ser Ala Ala Ser Ser Ser Ala Ala Ser
1 5 10 15
Ser Ser Ala Ala
20
One of dna sequence dna of this aminoacid sequence is SEQ ID NO:4
tcctcctccg ccgcctcctc ctccgccgcc tcctcctccg ccgcctcctc ctccgccgcc 60
The invention effect
The present invention is when making up people's Flt3 part and human IgG1's the segmental fusion gene of Fc, by artificial redesign Flt3 ligand gene and and the Fc fragment between joint sequence, the avidity of this fusion rotein and the expression amount in mammalian cells such as CHO thereof are significantly improved.
The following example is for better explanation the present invention, is not in order to limit the invention in the scope defined in the claim.
Embodiment
Embodiment 1
The preparation of total length reorganization Flt3 ligand gene
The artificial sequence of Flt3 ligand gene
According to mammalian cell access to your password the son preferences, the segmental aminoacid sequence of known Flt3 part (SEQ ID NO:2, GenBank accession number T03858), designed one section new reorganization Flt3 ligand gene (SEQ ID NO:3):
acccaggact gctccttcca gcactccccc atctcctccg acttcgccgt gaagatccgc 60
gagctgtccg actacctgct gcaggactac cccgtgaccg tggcctccaa cctgcaggac 120
gaggagctgt gcggcggcct gtggcgcctg gtgctggccc agcgctggat ggagcgcctg 180
aagaccgtgg ccggctccaa gatgcagggc ctgctggagc gcgtgaacac cgagatccac 240
ttcgtgacca agtgcgcctt ccagcccccc ccctcctgcc tgcgcttcgt gcagaccaac 300
atctcccgcc tgctgcagga gacctccgag cagctggtgg ccctgaagcc ctggatcacc 360
cgccagaact tctcccgctg cctggagctg cagtgccagc ccgactcctc caccctgccc 420
cccccctggt ccccccgccc cctggaggcc accgccccca ccgccccc 468
Wherein 69 Nucleotide of this gene 5 ' end are the signal peptide sequences of being convenient to the mammalian cell secretory protein.
The aminoacid sequence of this product is SEQ ID NO:2
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30
Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp
35 40 45
Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala
50 55 60
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His
65 70 75 80
Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95
Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu
100 105 110
Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125
Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140
Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
145 150 155
Add the Flt3 part recombination of top connection and the gene design that joint merges
This gene 3 '-end adds top connection (Ser 3Ala 2) 4The coding region, its encoding sequence is: 5 '-tcctcctccgccgcctcctc ctccgccgcc tcctcctccg ccgcctcctc ctccgccgcc-3 ' (SEQ ID NO:4).
The brand-new gene of this design and recombination and known human IgG1's Fc fragment (Genbank accession number P01857) are merged, form new Flt3 ligand gene/joint/IgG1 Fc gene.
Embodiment 2
The synthetic of the full Flt3 ligand gene of natural and artificial design
Respectively there are 20 base complementrity districts to split into the oligonucleotide sequence of 16 length according to each dna fragmentation two ends the Flt3 ligand gene sequence of above-mentioned artificial design, natural Flt3 ligand gene sequence and their complementary strand sequence, joint sequence at 83-86bp, after carrying out the PAGE purifying, carry out PCR according to following method.
Reaction system:
Reagent name quantity (Tl)
10XPCR damping fluid 5
Mg ++(25 mmoles/liter) 2.5
DNTP (10 mmoles/liter) 2
Taq archaeal dna polymerase (5T/Tl) 1
Oligonucleotide (100T mol) each 1
H 2O to 50 microlitre
Cycling condition:
Pre-sex change: 94 ℃ 2 minutes
Major cycle: 94 ℃ 1 minute, 52 ℃ 1 minute, 72 ℃ 1 minute, circulate 30 times.
Extend the back: 72 ℃ 5 minutes.
After PCR finishes, on 1% agarose electrophoresis, identify the fragment of amplification, discovery has a unique band in molecular weight 450-500bp scope, pGEM-T carrier (Promega company is cloned into according to ordinary method in glue recovery test kit recovery back with Promega company, Madison, MI) on, transformed into escherichia coli DH5 α bacterial strain, select 5 candidate clones after blue hickie screening of process and enzyme are cut evaluation then, to wherein two carry out dna sequencing, according to sequencing result, confirm that one of them sequence is entirely true.
Natural Flt3 ligand gene note is made Flt3/N, the Flt3 ligand gene note of artificial design is made Flt3/M, by above-mentioned PCR work, four fusion genes of the Flt3/N-IgG1Fc of the similar Flt3/M-IgG1Fc that synthesizes artificial design respectively, Flt3/M-joint-IgG1Fc, natural encoding sequence and Flt3/N-joint-IgG1Fc.
Embodiment 3
The structure of expression vector
Cut 4 kinds of antigen-4 fusion protein gene clones' that obtain among the embodiment 2 DNA with Xba I and Nhe I enzyme, and plasmid pMG18 (DEVELOPMENT OF TOOLS FOR ENVIRONMENTALMONITORING BASED ON INCP-9 PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, C.M.Thomas School of Biological Sciences, University ofBirmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept ofMicrobiology, Belarus State University Scorina Av.4, Minsk 220080 Belarus), its plasmid map such as Fig. 1.
Reclaim with carrying out glue after the separation of 1% agarose gel electrophoresis, the DNA of gained purifying is cloned on the pMG18 carrier according to a conventional method, transformed into escherichia coli DH5 α bacterial strain.Pass through two that blue hickie screens and enzyme is cut in 5 candidate clones selecting after the evaluation and carry out dna sequencing,, confirm that one of them sequence is entirely true according to sequencing result.
Use aforesaid method, made up 4 kinds of carriers, express Flt3/M-IgG1Fc, the Flt3/M-joint-IgG1Fc of artificial design, Flt3/N-IgG1Fc and four kinds of fusion genes of Flt3/N-joint-IgG1Fc of natural encoding sequence respectively.
Embodiment 4
The transfection of Chinese hamster ovary celI and the screening of recombinant clone
The e.colistraindh5 inoculation that will transform as 4 kinds of expression vectors that above-mentioned embodiment 3 makes up is respectively at increasing in 100 milliliters of LB substratum, with the UltraPure Plasmid DNAPurification Kit extracting and purifying plasmid DNA of Qiagen formula.Purification condition is according to manufacturers instruction.
Selecting to carry out continuous 9 all screening Tetrahydrofolate dehydrogenase activity and amicillin resistances on the substratum, cultivate in the enterprising limit by row dilution of 96 orifice plates then, carry out continuously 3 times, carry out mono-clonalization.Successively obtained 86 of Flt3/M-IgG1Fc clones, Flt3/M-joint-IgG1Fc clones 69,77 of Flt3/N-IgG1Fc clones, and Flt3/N-joint-IgG1Fc clones 91.
Embodiment 5
The research of fusion protein expression
Choosing monoclonal cell ties up on the RPM1641 substratum with 37 ℃, 5%CO 2In incubator, carry out amplification cultivation, centrifugal, measure its expression amount with ELISA.The average expression intensity that found that Flt3/M-IgG1Fc and Flt3/M-joint-IgG1Fc clone is apparently higher than Flt3/N-IgG1Fc and Flt3/N-joint-IgG1Fc clone.The results are shown in table 1.
The comparison of the expression amount of table 1 fusion gene
The structure of fusion gene Flt3/M- IgG1Fc Flt3/M-joint-IgG1Fc Flt3/N- IgG1Fc Flt3/N-joint-IgG1Fc
Expression amount (mg/litre) 3.14 3.22 0.87 0.72
From The above results as seen, the expression intensity of reorganization Flt3 ligand gene in Chinese hamster ovary celI through artificial design is higher about 4 times than natural gene.With Protein A nucleophilic chromatography and ion exchange chromatography, having obtained from the supernatant liquor of above-mentioned 4 kinds of expression of cell lines proves through SDS-PAGE electrophoretic examinations, and purity is greater than 92% recombinant protein.
The product of above-mentioned affinity chromatography has obtained the sample of purity＞98% once more through sieve chromatography.The sample of gained is used for following further research.
Embodiment 6
Avidity research
With 5 * 10 6Mouse WWF7 (expressing Flt3) cell is divided equally into 15 pipes, and according to 2,4,8, the amount of 16ug/ pipe joins in the cell with reorganization Flt3 ligand fusion protein to be measured (1.2 mg/ml), 37 ℃ of 5%CO 2Cultivate after 24 hours in the incubator and add MTT (tetramethyl-azo azoles) to final concentration 1 mg/litre, continuation cultivation 1 hour, the photoabsorption of on microplate reader, reading 570 nanometers.Hereinafter table 2 is experimental result (data are the percentage ratio that accounts for 16 microgram positive controls in the table).
Table 2 avidity relatively
The structure of fusion gene Positive control Flt3/M- IgG1Fc Flt3/M-joint-IgG1Fc Flt3/N- IgG1Fc Flt3/N-joint-IgG1Fc
2 micrograms 21.1 23.7 101.7 22.0 113.6
4 micrograms 49.6 46.2 266.2 50.7 301.4
8 micrograms 67.7 67.4 374.3 86.5 365.5
16 micrograms 100.0 103.8 467.6 99.7 456.7
The above results explanation relatively contains joint and does not contain the avidity of the fusion rotein of joint, and the adding owing to this fusion gene center tap sequence makes its avidity improve more than 5 times.
Sequence table
＜110〉the strong biotech research center of Shanghai Lan Sheng state
＜120〉reorganization Flt3 ligand gene, and fusion gene and product
<130>016012
<160>5
<170>PatentIn version 3.0
<210>1
<211>468
<212>DNA
＜213〉people (Homo Sapiens)
<400>1
acccaggact gctccttcca acacagcccc atctcctccg acttcgctgt caaaatccgt 60
gagctgtctg actacctgct tcaagattac ccagtcaccg tggcctccaa cctgcaggac 120
gaggagctct gcgggggcct ctggcggctg gtcctggcac agcgctggat ggagcggctc 180
aagactgtcg ctgggtccaa gatgcaaggc ttgctggagc gcgtgaacac ggagatacac 240
tttgtcacca aatgtgcctt tcagcccccc cccagctgtc ttcgcttcgt ccagaccaac 300
atctcccgcc tcctgcagga gacctccgag cagctggtgg cgctgaagcc ctggatcact 360
cgccagaact tctcccggtg cctggagctg cagtgtcagc ccgactcctc aaccctgcca 420
cccccatgga gtccccggcc cctggaggcc acagccccga cagccccc 468
<210>2
<211>156
<212>PRT
＜213〉people (Homo Sapiens)
<400>2
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30
Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp
35 40 45
Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala
50 55 60
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His
65 70 75 80
Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95
Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu
100 105 110
Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125
Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140
Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
145 150 155
<210>3
<211>468
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉Chong Xinsheji Flt3 part encoding sequence
<400>3
acccaggact gctccttcca gcactccccc atctcctccg acttcgccgt gaagatccgc 60
gagctgtccg actacctgct gcaggactac cccgtgaccg tggcctccaa cctgcaggac 120
gaggagctgt gcggcggcct gtggcgcctg gtgctggccc agcgctggat ggagcgcctg 180
aagaccgtgg ccggctccaa gatgcagggc ctgctggagc gcgtgaacac cgagatccac 240
ttcgtgacca agtgcgcctt ccagcccccc ccctcctgcc tgcgcttcgt gcagaccaac 300
atctcccgcc tgctgcagga gacctccgag cagctggtgg ccctgaagcc ctggatcacc 360
cgccagaact tctcccgctg cctggagctg cagtgccagc ccgactcctc caccctgccc 420
cccccctggt ccccccgccc cctggaggcc accgccccca ccgccccc 468
<210>4
<211>60
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉joint peptide-coding sequence
<400>4
tcctcctccg ccgcctcctc ctccgccgcc tcctcctccg ccgcctcctc ctccgccgcc 60
<210>5
<211>20
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉joint peptide
<400>5
Ser Ser Ser Ala Ala Ser Ser Ser Ala Ala Ser Ser Ser Ala Ala Ser
1 5 10 15
Ser Ser Ala Ala
20

Claims (8)

1. a reorganization Flt3 ligand gene sequence is characterized in that this Flt3 ligand gene has the nucleotide sequence of SEQ IDNO:3.
2. a fusion gene is characterized in that, human IgG1's antibody Fc fragment gene that this fusion gene contains reorganization Flt3 ligand gene sequence as claimed in claim 1 and merges with it.
3. fusion gene as claimed in claim 2, it is characterized in that, this fusion gene also comprises one section joint sequence that is positioned between reorganization Flt3 ligand gene and the human IgG1's antibody FC, and described joint sequence amino acid sequence coded is SEQ ID NO:5.
4. fusion gene as claimed in claim 3 is characterized in that, the dna sequence dna of described joint is SEQID NO:4.
5. a recombinant vectors is characterized in that, this recombinant vectors contains the described fusion gene of claim 2.
6. a recombinant host cell is characterized in that, this host cell contains the described carrier of claim 5.
7. a recombinant protein is characterized in that, this protein is the fusion rotein of Flt3 part and human IgG1's antibody FC, and described Flt3 part is the sequence encoding of the described reorganization of claim 1 Flt3 ligand gene.
8. recombinant protein as claimed in claim 7 is characterized in that, this protein also contains the joint peptide ammino acid sequence SEQ IDNO:5 between reorganization Flt3 ligand gene and human IgG1's antibody FC.
CN 01132280 2001-11-23 2001-11-23 Recombinant Flt3 ligand gene and its fusion gene and product Expired - Lifetime CN1234855C (en)

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